LAUSR

Hydrolysates are used as media supplements although their role is not well characterized. In this study, cottonseed hydrolysates, which contained peptides and galactose as supplemental substrates, were added to CHO batch cultures, enhancing cell growth, IgG titers, and productivities. Extracellular metabolomics coupled with TMT proteomics revealed metabolic and proteomic changes in cottonseed-supplemented cultures. Shifts in production and consumption dynamics of glucose, glutamine, lactate, pyruvate, serine, glycine, glutamate and aspartate suggest changes in TCA and glycolysis metabolism following hydrolysate inputs. Quantitative proteomics revealed 5521 proteins and numerous changes in relative abundance of proteins related to growth, metabolism, oxidative stress, protein productivity, and apoptosis/cell death at day 5 and day 6. Differential abundance of amino acid transporter proteins and catabolism enzymes such as BCAT1 and FAH can alter availability and utilization of several amino acids. Also, pathways involved in growth including the polyamine biosynthesis through higher ODC1 abundance and hippo signaling were upregulated and downregulated, respectively. Central metabolism rewiring was indicated by GAPDH downregulation, which corresponded with re-uptake of secreted lactate in the cottonseed-supplemented cultures. Overall, cottonseed hydrolysate supplementation modified culture performance by altering cellular activities critical to growth and protein productivity including metabolism, transport, mitosis, transcription, translation, protein processing, and apoptosis. Cottonseed hydrolysate, as a medium additive, enhances CHO cell culture performance Metabolite profiling and TMT proteomics characterize its impact on CHO cells Rewired nutrient utilization is observed via glycolysis, amino acid and polyamine metabolism Hippo signaling pathway impacts cell growth in the presence of cottonseed hydrolysate This article is protected by copyright. All rights reserved.